so processive that their run lengths and times were
similar to each other in standard assay buffer,
where microtubule length and imaging duration
become limiting (Fig. 2, C and D). However,
when assayed in high–ionic strength buffer, which
decreases dynein’s processivity (17), the 7D ensemble was more processive than the 4D ensemble (Fig. 2, C and D, and figs. S4 and S5).

We performed a similar analysis of kinesin
alone and kinesin ensembles on the chassis with
one, two, four, or seven motor-attachment sites
(1K, 2K, 4K, and 7K, respectively; Fig. 2, E to
H). The average velocities of the kinesin ensembles remained constant (Fig. 2F and fig. S6A),
whereas run lengths and times increased with increasing motor number (Fig. 2, G and H, and fig.
S6, B and C).

Recent models of motor ensemble behavior
using a transition-state framework predict run
lengths that are several orders of magnitude higher
than what we observed (18). In contrast, our data
suggest that motor microtubule binding dynamics
may be influenced by the presence and number of
other motors on a shared cargo, similar to previous
work (19–22). For one to seven kinesins or one or
two dyneins, velocity was unaffected by motor
number. However, for 4D and 7D ensembles,
velocity was decreased, suggesting that intermotor interference can affect motor stepping rate. To
test this hypothesis, we engineered the chassis
with locations for inactive mutant dyneins (denoted
dI) incapable of binding adenosine triphosphate
(ATP) at dynein’s main site of ATP hydrolysis;
this mutant binds microtubules tightly, but does
not move (23). Dynein ensembles programmed